PROJECT SUMMARY Diffuse large B-cell lymphoma (DLBCL), the most common aggressive type of lymphomas, is characterized by marked clinical and pathological heterogeneity that is reflected at the molecular level. It is well known that although the incidence rates for DLBCL were lower for African Americans (AA) than for European Americans (EA), AA patients were diagnosed at a significantly younger age and have worse 5-year survival compared with EA patients. The reason for this apparent racial/ethnic difference is unclear and cannot be fully explained by social stressors or access to care. We speculate that epigenetic variations could underlie the racial/ethnic differences because the pathogenesis of DLBCL is strongly linked to perturbation of epigenetic mechanisms, and population-specific cytosine modifications are a fundamental feature between human populations. In addition, greater epigenetic heterogeneity is linked with more aggressive DLBCL and poorer survival outcomes. Most prior studies of DLBCL epigenetics have assessed 5-methylcytosines (5mC) in predominantly EA patients. No studies have evaluated 5-hydroxymethylcytosines (5hmC), an emerging stable and abundant modification with distinct gene regulatory and cellular functions, due mainly to technical limitations. Most importantly, no studies have assessed concurrently 5mC and 5hmC in both EA and AA DLBCL patients. Without an effective technique to obtain the complete landscape of modified cytosines, development of targeted epigenetic approaches to improve DLBCL disparities is unlikely. The objective of this Exploratory/Developmental study is to distinguish 5hmC from 5mC at the time of DLBCL diagnosis between AA and EA patients and evaluate their clinical significance. Our hypothesis is that the 5mC/5hmC signatures in tumor tissues differ between AA and EA patients and that these differences contribute to the well-known racial/ethnic differences in DLBCL risk and outcomes. The Specific Aims are to 1) distinguish 5hmC from 5mC in EA and AA patients and evaluate population-specific 5mC/5hmC loci; and 2) determine therapeutic response-associated 5hmC/5mC loci. Specifically, we will obtain tissues for 120 patients with DLBCL (60 AA and 60 EA) from the University of Chicago Lymphoma Biobank. We will combine an innovative technique, the Tet-assisted Bisulfite Sequencing (TAB-Seq) and the new Illumina EPIC array to accurately distinguish 5hmC from 5mC. We will perform an epigenome-wide scan to assess differences in 5hmC/5mC modification levels between AA and EA patients at presentation and also integrate both modifications to predict therapeutic response, defined as relapse within 24 months after treatment. This proposal addresses a critical research question in a highly innovative, cost-efficient, and timely manner, providing important data for the first time on the complete landscape of modified cytosines for racial/ethnic differences in DLBCL. The results are expected to have positive impact because it is possible that the identified epigenetic loci/biomarkers will provide new targets for individualized preventive and therapeutic interventions to decrease mortality and burden of DLBCL.